Method of stabilizing enzymes

ABSTRACT

THIS INVENTION RELATES TO AN IMPROVED METHOD OF STABILIZING ENZYMES BY BONDING THE ENZYMES TO INORGANIC CARRIERS IN THE PRESENCE OF THEIR SUBSTRATES WHEREBY THE ENZYMES BECOME INSOLUBILIZED; AND THE PRODUCT FORMED BY THE METHOD.

May 30, 1972 R. A. MESSING METHOD OF STABILIZING ENZYMES Filed Oct. 14,1968 lill mem 02.12;

@ZPO/mm l NVEN TOR. Ralph A. Mess/ng MKM A T TORNE Y United "StatesPatent Clce 3,666,627 Patented May 30, 1972 v Y 3,666,627 METHOD FSTABILIZING ENZYMES Ralph A. Messing, Horseheads, N.Y., assignor tovCorning Glass Works, Corning, N.Y. Filed Oct. 14, 1968, Ser. No.767,116 Y Int. Cl. C07g 7/02 U.S. Cl. 195-68 4 Claims ABSTRACT 0F THEDISCLOSURE This invention relates to an improved method of stabilizingenzymes by' bonding the enzymes to inorganic Carriers in the presence oftheir substrates whereby the enzymes become insolubilized; and theproduct formed by the method.

-An enzyme is generally considered a biological catalyst capable ofinitiating, promoting, and governing a chemical reaction without beingused up in the process or becoming part of the product formed. It is asubstance synthesized `by plants, animals, some viruses andmicroorganisms. All enzymes isolated thus far have been found to beproteins, i.e.peptide polymers of amino acids. An enzyme may containprosthetic groups such as avin adenine dinucleotide, porphyrin,diphosphopyridine nucleotide, etc. Most enzyme are macromolecules,generally, having a molecular weight greater than 6,000.

The specicity of enzymes and their ability to catalyze reactions ofsubstrates atlow concentrations have been of particular interest inchemical analyses. Enzyme catalyzed reactions have been used for sometime for the qualitative and quantitative determination of substrates,activators, inhibitors, and also enzymes themselves. Until recently, thedisadvantages arising from the use of enzymes have seriously limitedtheir utility. Objection to the use of enzymes has been theirinstability, since they are susceptible to all the conditions whichnormally denature proteins, e.g. high temperature, concentrationdependence, pH changes,microbial attack, and autohydrolysis.Furthermore, vthe cost of large amounts of enzymes has made their use inroutine chemical analyses impractical.

Attempts have been made to prepare enzymes in an immobilized formwithoutloss of activity so that one sample could be used continuously for manyhours. The immobilized enzymes perform With increased accuracy all theoperations of ordinary soluble enzymes; that is, they can be used todetermine the concentration of a substrate, of anenzyme inhibitor, or ofan enzyme activator. These have been madeby physically entrappingenzymes in starch gel, polyacrylamide gel, agar, etc. Enzymes have beeninsolubilized by diazotizing them to cellulose derivativesand topolyaminostyrene beads. Enzymes have also beeninsolubilized onpolytyrosyl polypeptides and collodion matrices.y 'Ihe maindisadvantages of using such organic materials are (a) that they aresubject to microbial attack resulting from the presence of carbon atomsin the polymer chain `whereby the carrier is broken down and the enzymesolubilized; (-b) substrate diffusion in many cases becomes thelimitingy factor in reaction velocity thereby decreasing apparent enzymeactivity; and (c) when employed in chromatographic columns, the pH andsolvent conditions increase or decrease swelling which affects tlowrates of the substrate through the column.

My copending application, Ser. No. 702,829, now U.S. Pat. No. 3,556,945,led Feb. 5, 1968, describes a method of making stabilized enzymes bycontacting an aqueous solution of an enzyme having available aminegroups with an inorganic carrier, having a high surface area andreactive silanol groups, at up to room temperature or below and v forasuicientlperiod. of timeY for substantial bonding A* of the enzyme. Bythat process the enzyme is assumed to be coupled directly to the carrierby means of both hydrogen bonding and amine-silicate bonding. However, alimitation of the process is that a loss of activity may occur whenthere is bonding at the active sites on the enzyme molecule.

Quite surprisingly, I have discovered a method of lbonding enzymes toinorganic carriers which eliminates or substantially reduces the loss ofenzyme activity. This method involves bonding the enzyme to the carrierin the presence of its substrate and thus apparently blocking the activesites of the enzyme to avoid reaction of these sites with the carrier.By my improved process, highly stable enzymes can be prepared. Thesestabilized enzymes ind considerable use in analytical chemistry and mayalso be used in the preparation of chemicals, pharmaceuticals,foodstuifs and the detergent industry.

As used herein with reference to enzymes the terms stabilized,insolubilized, and immobilized have the following meaning. The termstabilized means a decrease in loss of enzyme activity as a function oftime and/or temperature. Insolubilized refers to substantially waterinsoluble and results from the coupling of the enzyme by chemical bondsto the insoluble inorganic carrier. Finally, immobilization is used tomean entrapment of the enzyme in a polymeric lattice or a semi-permeablemembrane.

In accordance with the present invention, I have discovered an enzymecomposite comprising an inorganic carrier having available oxide and/ orhydroxyl groups, an enzyme bonded to the carrier, and a substrate of theenzyme. Furthermore, my invention involves a method of making aninsolubilized enzyme composite by combining an inorganic carrier havingavailable oxide and/or hydroxyl groups with a substrate for an enzymeand reacting the enzyme with the carrier-substrate combination in anaqueous medium. The presence of the substrate serves to block the activesite of the enzyme during bonding to prevent reaction -with the carrier.

Enzymes capable of being stabilized as described herein include a widevariety of enzymes which may be classiied under three general headings:hydrolytic enzymes, redox enzymes, and transferase enzymes. The rstgroup, hydrolytic enzymes, in clude proteolytic enzymes which hydrolyzeproteins, e.g., papain, iicin, pepsin, trypsin, chymotrypsin, bromelin,keratinase, carbohydrases which hydrolyze carbohydrates, e.g. cellulase,amylase, maltase, pectinase, chitinase; esterases which hydrolyzeesters, e.g. lipase, cholinesterase, lecithinase, alkaline and acidphosphatases; nucleases which hydrolyze nucleic acid, e.g. ribonuclease,desoxyribonuclease; and amidases which hydrolyze amines, e.g. arginase,asparaginase, glutaminase, and urease. The second group are redoxenzymes that catalyze oxidation or reduction reactions. These includeglucose oxidase, catalase, peroxidase, lipoxidase, and cytochromereductase. In the third group are transferase enzymes that transfergroups from one molecule to another. Examples of these areglutamicpyruvic transaminase, glutamic-oxalacetc transaminase,transmethylase, phosphopyruvic transphosphorylase. It should be notedthat the enzyme can be used alone or in combination with other enzymes.

The carriers are inorganic materials having available oxide and/orhydroxide groups. These materials must be substantially water insolubleand are either weak acids or weak bases. They may also be classified interms of chemical composition as siliceous materials or non-siliceousmetal oxides. Of the siliceous materials, a preferred carrer is porousglass either in particulate form or as an integral piece such as a disc.Glass has the advantage in that it is dimensionally stable and thatitcan be thoroughly cleaned to remove contaminants as for example bysterilization. Porous glass useful as a carrier is readily available andsold commercially by Corning Glass Works as Code 7930 porous glass. Suchporous glass can be prepared having various pore dimensions inaccordance with the teachings of Hood et al., U.S. Patent No. 2,106,764,Chapman et al., U.S. patent application Ser. No. 565,372, now U.S.Patent No. 3,485,687 and W. Haller, U.S. patent application Ser. No.507,092, now U.S. Patent No. 3,549,524. Other siliceous inorganiccarriers which can also be used include colloidal silica (commerciallyavailable under the trademark CAB-O-SIL), wollastonite (a naturaloccurring calcium silicate), dried silica gel,

and bentonite. Representative non-siliceous metal oxides include aluminaand hydroxyapatite. These representative inorganic carriers may beclassiiied as shown in the table below:

INO RGANIC CARRIE RS In order to form the highly insolubilized enzymeand to prevent loss of enzyme activity, a substrate of the particularenzyme must be present during the bonding procedure. The importance ofthis may be illustrated in the bonding of trysin wherein by forming anenzymesubstrate complex prior to the carrier, the amino groups in theactive sites, i.e. nitrogen in the histidine residue, is occupied by thesubstrate. Masking these groups prevents reaction between the activesites of the enzyme and the carrier and leaves the sites available forfuture enzymatic rctions. The substrate additionally functions as acushion to prevent deformation of the enzyme` molecule and as acomplexing agent to reinforce the internal bonds of the enzyme. It isessential that the substrate be tailored to the specific enzyme. Theamount of substrate present during bonding should be suilicient toprotect the enzyme during bonding. Usually an equal amount by weight orenzyme to substrate may be used as a rough approximation. Substrates canbe used alone or in combination as long as they are compatible. But whentwo or more different types of enzymes are to be bonded, best resultswill be obtained when a substrate for each type of enzyme is present.Substrates for specic enzymes are shown in the table below.

TABLE OF ENZYMES AND RECOMMENDED PROTECTIVE SUBSTRATES SubstrateEnzyme 1. Proteolytic:

Papain, nein, pepsin, trypsin,

Cassin, gelatin, hemoglobin,

chymotrypsin bromelin, alkaline albumin, etc. protease. 2.Carbohydrases:

(a) Cellulase Cellulose, carboxymethyl cellulose. (b) Amylase- Starch.(c) Maltese--- Maltese. (d) Pectinase. Pectin.

- Chitosan, chitin, chitin nitrate.

(a) Glutamin pyruvie transaminase Glutamic acid.

Y (b) Phosphopyruvic transphospherylase-- Phosphopyruvie acid.

The accompanying drawing, which is a ilow sheet of the novel process,while not intended as a denition essentially illustrates the invention.A full discussion is sert forth hereinbelow. y

Referring now to my improved method of stabilizing the enzyme, in oneembodiment designated as Method I the carrier may be as formed bodies oras particulate materials. The carrier is rst combined with the substrateusually in an aqueous suspension or solution. Temperature is notcritical, but room temperature or slightly above is recommended. Thetime depends on the substrate and the temperature and generally lowmolecular weight substrates and higher temperatures result in shortercombining times', whereas higher molecular weight substrates and lowertemperatures result in longer combining times. The term combining isbroadly used to include saturating, coating, reacting, complexing,mixing, and dispersing. After the carrier and substrate have beencombined, excess strate may be removed.

In the next step, the enzyme in the presence of its sub.

strate is reacted with the carrier. Actually, the reaction may beconsidered in two parts: initially the enzyme reacts with the substrateand thereafter the available residues of the enzyme not reacting withthe substrateV are permitted to react with carrier. Generally, theenzyme in aqueous solution is added to the carrier-substrate com'-bination. The temperature of the reaction should be very low preferablyabout 5 C. or below. The pH at which the reaction is conducted is alsoimportant and it is recommended that the pH be either higher or lowerthanl the optimum pH of the enzyme substrate reaction. The

reaction should be allowed to continue for a sucient time, typically atleast 20 minutes, to bond the enzyme to the carrier. The product, bondedenzyme inan aqueous medium, may be stored and used as desired.

However, for most purposes the excess enzyme is re?I moved by filtrationor centrifugation and washing' in disltilled water. Finally, the bondedenzyme is dried by conventional techniques such as by drying in air orvacuum,v

spray drying, drum drying, and freeze drying. The dried bonded enzymemay also be stored for use.l

As an alternative procedure for stabilizing the enzyme, designated asMethod Il, the carrier is in the form of line` particulate materialpreferably having a maximum'par-V ticle size of ISU microns. The carrieris initiallycom# point that the enzyme and the carrier arecoprecipitated the addition of a precipitating agent.Coprecipitationrnay be either by dehydration or charge neutralization-`1n de.,

hydration, an organic solvent such as' acetone lor an alco` hol acts asthe precipitating agent. Charge 'neutralization results from theaddition of a saltsolution, elg.v ammonium sulfate and sodium sulfate,to neutralize the charge on the protein molecules and thecarrierlparticlesi The temperature during coprecipitation should, begenerally below room temperature. Finally, the precipitating vagent isremoved by such conventional procedures Vas ltratio'n, centrifuging,washing, and air drying. The product rob-` tained is a dried stabilizedenzyme composite'jwhich'is initially water insoluble. :i

My invention is further illustrated by 'the following examples: Y

,EXAMPLE i( A quantity of powderedporous 9,6% silica glass (595 A. poresize, -140 mesh U.S. ,Standard kSievey..'was washed in 0.2 N HNO3 withcontinuous sonication'for at least 30 minutes. The glass was washedseveral times with distilled water and heat cycled to 625 C. under anO2. y.stream for 30 minutes. Thereafter, the glass was cooled in the O2atmosphere. A 0.2% gelatin substrate solution was prepared by adding 100ml. distilled water to 200 mg. of gelatin (U.S.P. granular, 270 Bloom)and dissolving with heat and sonication.

To 500 mg. of porous glass was added 5 ml. of the gelatin solution andthe mixture agitated at 37 C. for 11/2 hours. The residual gelatinsolution (3.6- ml.) was separated from the glass by decantation.

The gelatin protected glass sample was cooled to 5 C. in a water bathand then 5 ml. otan aqueous solution containingY 1.39 ing/ml. ofalkaline protease from B. Subtilis (commercially available as Alcalase)was added. The enzyme was' allowed to react with the glass at 5 C. for171/2 hours. The enzyme solution was removed by ltration and theglasspa'rticles were thoroughly washed with water and air dried. A

A control sample was also prepared in which 500 mg. ofporousV glass wasadded to 1.4'ml. of distilled water to approximater the liquid volumeremaining in the gelatin protected sample after.decantation..Thereafter,the bonding procedure was similar to that of the sample in which theenzymewas bound inthe presence of its substrate.

The enzyme solutions and washes for each sample were analyzed forunbound protein V at a Wave length of 280 ma. The Weights of the reactedglass and the amounts of enzyme bound (bydilerencearttZSO ma) were asfollows:

Enzyme 'Dried protein Enzyme, Sample sample, g. bound, mg. Ing/g.

Substrate protected 1.023 2. 36 2. 31 Control 1. 020 2. 78 2. 73

The bound enzyme was thenv assayed as follows:

(A) ANSON HEMOGLOBIN (pH 9.72, MINUTES, 37 C.)

Active Mg. E activity] enzyme, Sample g. sample percent Substrateprotected. 1. 32 72 Control O. 92 30 (B) AZOCOLL (pH, 10, 15 MINUTES, 37C.)

Active yMg. E activity] enzyme, Sample g. sample percent These resultsclearly show that the substrate protected system hasv substantiallygreater activity than the control and, therefore, the substrateprotected system reduces the loss of enzymatic activity during bondingof the enzyme to an inorganic carrier.

EXAMPLE II Following the procedure of Example I, 200 mg. of the alkalineproteaseenrzyme was `bonded to 600 mg. of Aamorphous colloidal 'silicaparticles (CAB-OEIL) in the presence and absence of 200 mg. of theprotective gelatin substrate. The bound samples were assayed with ahemoglobin substrateV at pH 9.7 .with 1,0 minutes and 45 minutesincubations at 55?' C. The results were as follows:

The increased recovery of the bound enzyme relative to the free enzymeat the longer incubation time (45 minutes) is due to the gradual releaseof enzyme into solution with time while the enzyme in free solution isdestroyed by denaturation.

6 EXAMPLE nr Following the procedure of Example l, 0.6 gram of bentonitewhich had been heated to 500 C. in O2 for 11/2 hours was added to l0 ml.of la 2% gelatin solution. After the carrier and substrate werethoroughly mixed, 10 ml. of a 2% (200 mg.) alkaline protease solutionwas added at room temperature. The mixture of carrier, substrate, andenyzme was cooled to 5 C. and allowed to react overnight. The productwas ltered on a Bchner funnel, washed with distilled water, and airdried. The enzyme protein bound to the bentonite was determined from theresidual protein (spectrophotometrically at 280 ma) in the wash and theltrate. A control sample was prepared by adding 10 ml. of the 2% (200mg.) alkaline protease solution to 1.0 g. of bentonite and allowing themixture to react overnight. The control was then treated in the samemanner as the substrate protected sample.

The Weight of the reacted bentonite and the amount of enzyme bound wereas follows:

Mg. enzyme Mg. enzyme] Sample Weight, g. protein bound g. sampleSubstrate protected 0. 6870 86 125 Control 1. 1244 146 130 The boundenzyme was then assayed by Anson Hemoglobin (pH 9.75, 10 minutes, 37C.).

Active Mg. E activity] enzyme,

Sample g. sample percent Substrate protected 40. 0 32 Control 1. 2 0.9

EXAMPLE 1V Mg. enzyme Mg. enzyme] Sample Weight, g. protein bound g.sample Substrate protected 0.6026 54 90 Control 1. 0435 88 84 The boundenzyme was then assayed by Anson Hemoglobin (pH 9.75, l0 minutes, 37C.).

Active Mg. E activity] enzyme,

Sample g. sample percent Substrate protected 7. 0 7. 8

Control 1. 2 1. 4

EXAMPLE V Following the procedure and using the same amount of reagentsof Example III, a substrate protected and a control sample were preparedusing hydroxyapatite (Ca1o(OH)2(PO4)6 Baker Reagent Grade) v as thecarrier. v

The weight of the reacted hydroxyapatite and `the amount of enzyme boundwere as follows:

Mg. enzyme Mg. enzyme] Sample Weight, g. protein bound g. sampleSubstrate protected 0.6455 58 90 Control 1. 008 99 98 The bound enzymewas then assayed by Anson Hemoglobin (pH 9.75, 10 minutes, 37 C.).

{Following the procedure of Example I, a quantity of powdered porous 96%silica glass (790150 A. pore size, 100 mesh U.S. Standard Sieve) wascleaned and prepared for bonding. A 3 dextrose solution was prepared bydissolving 3 g. dextrose monohydrate to 100 ml. of water. To 4.0 g. ofporous Iglass in a test tube was added 8.0 ml. of the dextrose solutionand the tube was placed in a Water bath at C. After the sample hadcooled, 8.0 ml. of precooled glucose oxidase (45.5 units/mg., 5 mgJ m1.)was added and the mixture was allowed to react for 21V2 hours at 5 C.The glass was separated by filtration and washed with Water. The enzymesolution and washes were analyzed for unbound protein at 280 ma. Byprotein dilerence the amount of enzyme bound to the Aglass was 5.5 mg.enzyme/g. sample.

A control sample was prepared in the same manner as the substrateprotected sample with the exception that 8.0 ml. of distilled Water wassubstituted for the dextrose solution. The amount of enzyme bound to theglass was 4.6 mg. enzyme/ g. sample.

The bound glass samples were then assayed by incubation at roomtemperature in 0.01 M phosphate buffer, pH 7.0, using 22.4 mg.beta-D-glucose per ml. buier. To determine the H2O2 produced, a 2.5 ml.aliquot of reaction mixture was removed and 0.025 ml. of 1%o-dianisidine in methanol was added followed by 0.5 ml. of peroxidase(0.04 mgJml.) solution. A unit of activity is equivalent to theproduction rate of 1 micromol H2O2 per hour at room temperature. TheH2O2 produced is determined by its decomposition in the presence ofperoxidase and o-dianisidine which upon oxidation increases the opticaldensity at 460 mp. The results of the assay were as follows:

(A) INITIAL ACTIVITY Active enzyme, percent (B) AFTE R 24HOU'RS LEACHINGAT ROOM TEMPERATURE Active enzyme, percent Mg. activity/g. Sample sampleSubstrate protected Control 0, 57

EXAMPLE VII To 200 g. of bentonite were added with stirring 2.0 litersgelatin solution (70 g. of gelatin dissolved in 2 liters H2O) at 80 C.The stirring was continued while the Vtemperature was allowed to drop to39 C.`Then 1.0 liter of alkaline protease solution at 5 C. (60 g. ofalkaline proteasein 1.0 literof H2O) was added with stirring andimmediately 9.0 liters of acetone were added. The temperature fell to`19" C. After the mixture was stirred and permitted to settle, it wasltered and washed with acetone. The dried filtrate gave a yield of 297.5g.

The product was assayed by vthe following procedures:

(A) ANsoN HEMOGL'OBTN (pH 9.75, avec., 1o MIN.)

. Active Mg. enzyme/g. enzyme, Sample sample percent Substrate protected91 (E) lizocoLL 1N DETEEGENT SOLUTION tasje., 1o ML., so

MG. AzoooLL) v i Y s Substrate protectedl '.y

(o) AzocoLL 1N DETERGENT SOLUTION 1379's., 25 MG.

AzocoLL, 1o MLN.)

` Active Sample sample percent Substrate protected 283 I: p ',142

It was noted that the coprecipitation technique was particularlyeffective. The fact thattheactive enzyme appears to give activity valuesgreater than may be explained by the fact thatth VsubstrateVpfotctedibo'nd samples has increased "thermal stability at higher tern-lperatures as compared to the' free unbound enzyme.'

It will be apparent to'those skilled in theart that many` variations andmodifications of. the invention as .herein-v above set forth may be madewithout departing from. the spirit and scope of the invention. Theinvention is not limited to those details andV applications described,except as set forth in the appended claims.

I claim:

1. A method of preparing an insolubilized enzyme of increased enzymaticactivity which comprisesthe steps of:

(a) combining a substrate of the enzyme Ywith a porousA glass carrier;and, v Y ,v

(b) contacting a solutionof s the enzyme ,with .the

carrier-substrate combination.

2. 'I'he method, as claimed in claim 1, wherein the contact of step (b)is carried out at a'temperature below about 5 C. f i" 3. The method,'asc'laimed in"claim"'1,"wherein the enzyme insolubilized isatproteaseJa-.iv` H I 4. The method, as claimed infclaim 1,1 wherein theenzyme insolubilized is glucose oxidase? i r References Citevdfzz.` .le

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